3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic acid] hexasodium salt AND 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6)

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

lemna species

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2016 to 31 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: at 0 and 100 mg/L on day 0, 2 and 4 from fresh solutions, on day 2, 4 and 7 from old solutions (range finding at 1, 10 and 100 mg/L on day 0, 3 and 7)
- Sample storage conditions before analysis: frozen

Vehicle:

no

Details on test solutions:

directly dissolved in culture medium

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Strain: Lemna minor
- Source (laboratory, culture collection): Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada.

ACCLIMATION
- Acclimation period: at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) during at least 7 days
- Culturing media and conditions (same as test or not): yes (Swedish Standard (SIS) Lemna growth medium)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Remarks on exposure duration:

renewal at day 2 and 4

Test temperature:

24 ± 1 ºC

pH:

in fresh solutions: 6.7-6.9
in old solutions: 7.1-7.9

Nominal and measured concentrations:

nominal:
pre-test: 0, 1, 10 and 100 mg/L
main test: 0, 1.0, 3.2, 10, 32 and 100 mg/L
measured:
pre-test: on day 0, 3 (old) and 7 (separate sample without lemna): 77-100%
main test: in fresh and old solutions: controls < LOQ; at 100 mg/L 97-111%

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks (500 mL) each containing 250 mL of test preparation
- Aeration: no
- Agitation: no
- Renewal rate of test solution: main study static test (pre-test semistatic with renewal on day 3)
- No. of fronds per colony: 3 colonies (9 fronds)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, Swedish Standard (SIS) Lemna growth medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continously
- Light intensity and quality:ca 7000 lux

EFFECT PARAMETERS MEASURED
- Determination of frond number: yes on days 0, 3, 5 and 7
- Determination of biomass: dry weight
- Determination of frond area: no
- Other: frond size, appearance, root length and number of colonies present, calculation of growth rate and yield

RANGE-FINDING STUDY
- Test concentrations: 0, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes (effect on growth (10-17%) at 1.0, 10 and 100 mg/L)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: measured concentrations 97-111% of nominal

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: measured concentrations 97-111% of nominal

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: measured concentrations 97-111% of nominal

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: measured concentrations 97-111% of nominal

Details on results:

- Any visual signs of phytotoxicity (abnormalities): at 0, 1.0, 3.2 and 10 mg/L all fronds appeared green, at 32 and 100 mg/l some staining was observed
- Decrease in frond size: none observed
- root size: slightly decreased at 32 and 100 mg/L
- doubling time: 1.89 days


Statistical analysis of the yield data was carried out for the control and all test concentrations. There were no statistically significant differences between the control, 1.0, 3.2, 10, 32 and 100 mg/L test concentrations (P≥0.05). However, visual inspection shows there to be 23 and 25% inhibition of yield at 32 and 100 mg/L respectively and as such the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was considered to be 10 mg/L.

Results with reference substance (positive control):

Separate study with dichlorophenol at 0.625, 1.25, 2.5, 5.0 and 10 mg/L gave EC50 values between 3.0 and 3.4 for growth rate and 1.4 and 1.8 mg/L (frond number and dry weight)

Reported statistics and error estimates:

NA

Inhibition of Average Specific Growth Rate and Yield Based on Frond number

Nominal Concentration (mg/L)	Average Specific Growth Rate		Yield
	0 – 7 Day	% Inhibition	0 – 7 Day	% Inhibition
0	0.302±0.006	-	66±3	-
1.0	0.319±0.016	[6]	75±9	[14]
3.2	0.311±0.014	[3]	70±7	[6]
10	0.300±0.003	1	64±2	15
32	0.281±0.027	7	56±12	3
100	0.289±0.023	4	60±11	9

 

Inhibition of Average Specific Growth Rate and Yield Based on dry weight

Nominal Concentration (mg/L)	Average Specific Growth Rate		Yield
	0 – 7 Day	% Inhibition	0 – 7 Day	% Inhibition
0	0.309±0.012	-	5.7 ± 0.5	-
1.0	0.427± 0.010	[9]	7.5 ± 0.6	[32]
3.2	0.409± 0.015	[5]	6.6 ± 0.7	[16]
10	0.396± 0.004	[2]	6.0 ± 0.2	[5]
32	0.353± 0.037	9	4.4 ± 1.2	23
100	0.350± 0.030	10	4.3 ± 1.0	25

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate for the substance is > 100 mg/L
All EC50 values deroved were > 100 mg/L

Executive summary:

Lemna minor was exposed to an aqueous solution of the substance at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days (semistatic with renewal on day 2 and 4), under constant illumination at a temperature of  24 ± 1 °C.  The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development. Measured concentrations were 97 -111% of nominal. No effects on yield and growth rate were observed at any of the concentrations tested based on frond count and/or dry weight. The EC50 is > 100 mg/L.

Description of key information

Lemna minor was exposed to an aqueous solution of the substance at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for a period of 7 days (semistatic with renewal on day 2 and 4), under constant illumination at a temperature of  24 ± 1 °C.  The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development. Measured concentrations were 97 -111% of nominal. No effects on yield and growth rate were observed at any of the concentrations tested based on frond count and/or dry weight. The EC50 is > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

100 mg/L

Additional information

Lemna was chosen as test organism, because the substance is a colorant. In an algae study a colorant may interfere with the measurements of biomass.